Metformin attenuates alveolar bone destruction in mice with apical periodontitis and inhibits pro-inflammatory cytokine synthesis in lipopolysaccharide-stimulated RAW264.7 through the AMPK-mTOR-NF-κB pathway

Chunmei Ren¹Kento Tazawa^1*Nobuyuki Kawashima¹Risa Ohshima¹Yamato Okada¹Shihan Wang¹Ziniu Yu¹Peifeng Han²Yujin Ohsugi^3,4,5Sayaka Katagiri^4,5Takashi Okiji^1,6

• ¹Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Insitute of Science Tokyo, Tokyo, Japan
• ²Department of Endodontics, Affiliated Stomatology Hospital, Guangzhou Medical University, Guangzhou, China
• ³Section of Vascular Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, United States
• ⁴Department of Oral Biology, Graduate School of Medical and Dental Sciences, Insitute of Science Tokyo, Tokyo, Japan
• ⁵Section of Oral-Systemic Health, Oral Science Center, Institute of Science Tokyo, Tokyo, Japan
• ⁶Department of Endodontics, The Nippon Dental University, Tokyo, Japan

Introduction: Apical periodontitis, caused by bacterial infection through the root canals, is characterized by chronic inflammation and bone resorption around the root apex. Metformin, a firstline therapeutic drug for type 2 diabetes mellitus, has attracted attention for its potential antiinflammatory properties and role in regulating bone homeostasis. The hypothesis in this study was that metformin inhibits bone destruction in apical periodontitis by suppressing macrophage-mediated inflammatory responses. The aim of this study was to evaluate the effect of systemic metformin administration on experimentally induced apical periodontitis development in an animal model and clarify the underlying anti-inflammatory mechanism of metformin in lipopolysaccharide-stimulated mouse macrophages. Methods: Evaluations on the effects of metformin on the progression of periapical lesions were conducted in experimentally induced mouse apical periodontitis in vivo, and its anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages in vitro were analyzed. Results: Metformin significantly reduced periapical bone destruction on postoperative days 21 and 28, and decreased the number of osteoclasts on the periapical alveolar bone on postoperative day 28. It also suppressed pro-inflammatory cytokine expression and nuclear factor kappa B signaling in lipopolysaccharide-stimulated RAW264.7. RNA-sequencing data revealed the downregulation of the mammalian target of rapamycin signaling after metformin treatment, which was confirmed by the downregulation of the mammalian target of rapamycin phosphorylation by metformin. Furthermore, metformin activated adenosine monophosphate-activated protein kinase, a potent negative regulator of mammalian target of rapamycin complex 1. The suppression of inflammatory cytokine expression by metformin was abolished by compound C, a potent adenosine monophosphate-activated protein kinase inhibitor. Discussion: This study revealed that metformin suppressed inflammatory bone destruction in periapical lesions. The mechanism partially involves inhibiting the mammalian target of rapamycin/nuclear factor-kappa B signaling in macrophages through adenosine monophosphate-activated protein kinase signaling activation. Findings from this study show that metformin has therapeutic potential in inflammatory bone destruction, such as apical periodontitis.